The Comet Moment as a Measure of DNA Damage in the Comet Assay

Abstract

The development of rapid assays of radiation-induced DNA damage requires the definition of reliable parameters for the evaluation of dose—response relationships to compare with cellular endpoints. We have used the single-cell gel electrophoresis (SCGE) or ‘comet’ assay to measure DNA damage in individual cells after irradiation. Both the alkaline and neutral protocols were used. In both cases, DNA was stained with ethidium bromide and viewed using a fluorescence microscope at 516–560 nm. Images of comets were stored as 512 × 512 pixel images using OPTIMAS, an image analysis software package. Using this software we tested various parameters for measuring DNA damage. We have developed a method of analysis that rigorously conforms to the mathematical definition of the moment of inertia of a plane figure. This parameter does not require the identification of separate head and tail regions, but rather calculates a moment of the whole comet image. We have termed this parameter ‘comet moment’. This method is simple to calculate and can be performed using most image analysis software packages that support macro facilities. In experiments on CHO-K1 cells, tail length was found to increase linearly with dose, but plateaued at higher doses. Comet moment also increased linearly with dose, but over a larger dose range than tail length and had no tendency to plateau.